PMID:14672667
| Citation |
Spudich, GM, Miller, EJ and Marqusee, S (2004) Destabilization of the Escherichia coli RNase H kinetic intermediate: switching between a two-state and three-state folding mechanism. J. Mol. Biol. 335:609-18 |
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| Abstract |
Escherichia coli RNase H folds through a partially folded kinetic intermediate that mirrors a rarely populated, partially unfolded form detectable by native-state hydrogen exchange under equilibrium conditions. Residue 53 is at the interface of two helices known to be structured in this intermediate. Kinetic refolding studies on mutant proteins varying in size and hydrophobicity at residue 53 support a contribution of hydrophobicity to the stabilities of the kinetic intermediate and the transition state. Packing interactions also play a significant role in the stability of these two states, though they play a much larger role in the native-state stability. One dramatic mutation, I53D, results in the conversion from a three-state to a two-state folding mechanism, which is explained most easily through a simple destabilization of the kinetic intermediate such that it is no longer stable with respect to the unfolded state. These results demonstrate that interactions that stabilize an intermediate can accelerate folding if these same interactions are present in the transition state. Our results are consistent with a hierarchical model of folding, where the intermediate consists of native-like interactions, is on-pathway, and is productive for folding. |
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| Keywords |
Circular Dichroism; Enzyme Stability; Escherichia coli/enzymology; Hydrogen/metabolism; Kinetics; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Protein Denaturation; Protein Folding; Ribonuclease H/chemistry; Ribonuclease H/genetics; Ribonuclease H/metabolism; Thermodynamics |
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